Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation.
Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.
Sygusch
J
J
Département de biochimie, Faculté de Médecine, Université de Sherbrooke, Fleurimont, Québec, Canada.
Beaudry
D
D
Allaire
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D010450:Endopeptidases; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D006168:Guinea Pigs; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation
D000596:Amino Acids; D007527:Isoenzymes; D010446:Peptide Fragments; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D010450:Endopeptidases; D005634:Fructose-Bisphosphate Aldolase